Reducing pain, inflammation, and fever with plant extracts (such as salicylate-containing plant extracts) can be traced throughout written human history. One hundred and fifty years ago, Felix Hoffman acetylated salicylic acid and created aspirin. Aspirin inhibits the cyclooxygenase (COX) enzymes COX-1 and COX-2, which synthesize inflammatory prostaglandins and thromboxanes. The ability to block inflammation via mediators such as prostaglandins and thromboxanes accounts for aspirin being the world's most used therapeutic agent.
Yet, it is still the challenge of the pharmaceutical chemist to develop more effective and less toxic agents to treat the signs and symptoms and development of acute inflammation as well as the long-term consequences of chronic inflammatory diseases Inflammation is a dynamic process with proinflammatory cytokines such as tumor necrosis factor (TNF)-a, interleukin (IL)-1b, and vascular endothelial growth factor (VEGF) playing central roles. A number of “biologicals” have been developed to treat inflammation, including agents that reduce the activity of specific cytokines or their receptors.
Neutralization of specific proinflammatory cytokines has “canonized” their causative role in inflammation and has changed the lives of millions of patients with these diseases. One drawback of anti-cytokine therapies is decreased host immune defense against infection and possibly cancer.
Many intracellular signaling molecules are involved in normal cellular functions and inflammation thus the effective concentration that does not elicit organ toxicity needs to be carefully determined.
Interleukin-1 (IL-1) is a potent pro-inflammatory cytokine that promotes chronic and acute inflammation, as well as harbors metabolic manifestations and is involved in hematopoiesis. IL-1 belongs to a large family of cytokines that consists of both pro-inflammatory and anti-inflammatory members; IL-1 receptor antagonist (IL-1Ra) is an anti-inflammatory IL-1 family member. IL-1alpha, IL-1beta and IL-1Ra, bind to the same receptor. The signaling receptor is IL-1R1. In order for signaling to occur, IL-1R1 requires a co-receptor, the IL-1R accessory protein (IL-1RAcP). Successful association with IL-1RAcP requires conformational changes that occur after IL-1 family members, IL-1α or IL-1β, bind to IL-1R1. However, binding to IL-1R1 at a higher affinity compared to IL-1α or IL-1β, IL-1Ra, IL-1Ra does not recruit IL-1RAcP, thus eliciting no inflammatory signal.
IL-1Ra may thus interfere with excessive IL-1 activities; IL-1 plays a critical role in many inflammatory diseases, such as gout, osteoarthritis and hypoxic tissue injury. However, IL-1 was also shown to be involved in non-classic inflammatory conditions, such as post-infarction cardiac failure, loss of beta cells during type 1 diabetes, elevated insulin resistance in type 2 diabetes and facilitation of tumor growth, angiogenesis and metastases. Both IL-1 alpha and IL-1beta are transcribed and translated as precursor proteins of 31 kDa. IL-1alpha can be found as a precursor or cleaved 17 kDa mature form. Precursor of IL-1alpha can be processed by the Ca2+-dependent protease calpain, a process which is not common in all cells. The necessity of IL-1alpha cleavage is not fully understood since both forms of the molecule are active and can trigger IL-1R1 signaling. IL-1beta, unlike IL-1alpha is not present in cells under normal conditions, and it is rapidly upregulated upon recognition of bacterial products by TLR signaling. Moreover, unlike IL-1alpha, IL-1beta precursor is inactive, and multiple steps control its activation. Transcription and translation of IL-1beta are not sufficient for its activity; a cleavage step of the precursor into active mature form is required. The classic cleavage of IL-1beta protein is executed by the cysteine protease caspase-1, which itself requires activation by a multi-protein complex, the inflammasome. IL-1beta can be secreted by secretory lysosomes, which contains the cytokine together with its activating protease, caspase-1 to the surrounding environment.
However, IL-1beta can be released due to cell death and loss of membrane integrity, either as mature or in its un-cleaved precursor form. Although IL-1beta can trigger IL-1R1 and promote inflammation and fever only in its mature form, the precursor of IL-1beta can be activated in the inflammatory site due to elevated levels of inflammatory proteases. Tt has been demonstrated that recombinant IL-1beta precursor can be cleaved by elastase, cathepsin G, collagenase, proteinase-3, mast cell chymase and Natural Killer (NK)/cytotoxic T cell (TCL)-granzyme A (Hazuda D J, Strickler J, Kueppers F, Simon P L, Young P R. Processing of precursor interleukin 1 beta and inflammatory disease. The Journal of biological chemistry. 1990; 265(11):6318-22. Epub 1990/04/15).
Hazude et al. also sequenced the processed proteins sequences and found the site of cleavage. According to their data, cleavage site can vary from Ile 103 to Asp 116. Using EL4 cells stimulation assay, it was shown that the cleavage products are active compared to the inactive precursor. In addition, it was shown that the IL-1beta precursor can be cleaved following incubation with synovial fluids from polyarthritis patients or bronchoalveolar lavage (BAL) from sarcoidosis patients, emphysema or infection after lung transplantation patients.
Not only neutrophil proteases appear to digest IL-1beta precursor, mast cell chymase was also shown to liberate the propeptide of IL-1beta, and to activate the cytokine. Chymase is a chymotrypsin-like serine protease present in secretory granules of mast cells. It is capable of promoting matrix degradation and inflammation. The 18 kDa IL-1beta product is indeed active, comparable to the mature IL-1beta. Natural killer (NK) and Cytotoxic T cells (TCL) enzyme, granzyme A, can also cut IL-1beta, and the site of cleavage was demonstrated to be following Arg 120, four residues C-terminally to the caspase-1 site (Asp 116).
Anakinra is a recombinant, non-glycosylated form of IL-1Ra was shown to relieve disease severity in various auto-inflammatory diseases, such as rheumatoid arthritis, gout, osteoarthritis and more (Dinarello C A, Simon A, van der Meer J W. Treating inflammation by blocking interleukin-1 in a broad spectrum of diseases. Nature reviews Drug discovery. 2012; 11(8):633-52. Epub 2012/08/02). Anakinra has been approved by the FDA since 2001 for the treatment of rheumatoid arthritis and recently for the treatment of cryopyrin-associated periodic syndromes (CAPS), a group of inherited auto-inflammatory syndromes in which excess IL-1 mediates exacerbated inflammatory events. Administered either locally or systemically, Anakinra is limited by an extremely short half-life and bares the risk of excessive systemic IL-1 blockade.
There is a need for more effective means of anti-IL-1 therapy for treating inflammation, such as by using IL-1Ra that is not constitutively active (as in the case of Anakinra), but is activated only at sites of excessive inflammation.